Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Chavez, Juan D.; Cilia, Michelle; Weisbrod, Chad R.; Ju, Ho-Jong; Eng, Jimmy K.; Gray, Stewart M.; Bruce, James E.

doi:10.1021/pr300041t

Cited by 62 publications

(104 citation statements)

References 79 publications

(272 reference statements)

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“…Virus-host protein complexes were enriched from the cryomilled tissue using sucrose density centrifugation as described in reference 16. Aphid transmission assays were performed on virus purified from plants infected with wild-type virus to verify that the virus was infectious and transmissible by aphids as described previously (16).…”

Section: Methodsmentioning

confidence: 99%

“…The BDP cross-linker dissolved in dimethyl sulfoxide was added to 100 to 500 g of partially purified virus to a final concentration of 1 mM. Concentration of BDP-NHP was selected based on workflows optimized in previous studies (16,19). Samples were incubated for 1 h at room temperature with constant mixing.…”

Section: Methodsmentioning

confidence: 99%

“…Such information is necessary for the development of novel virus management tools. However, the disordered nature of the RTP and its confinement to phloem has made protein interaction studies with PLRV and other related viruses challenging (16). In addition, molecular techniques such as yeast-two-hybrid and bimolecular fluorescence, which are traditionally used to probe protein-protein interactions (17), are not amenable to analyzing interactions that occur between multimeric protein complexes such as assembled virions, nor do they allow for the precise mapping of amino acids involved in the interaction without the generation of additional clones.…”

mentioning

confidence: 99%

“…Protein interaction reporter (PIR) technology has emerged as a powerful approach to probe in vivo and in vitro interactions between and within multimeric protein complexes (16,18,19). The strategy was developed to identify amino acid residues in interacting proteins that exist close to one another (typically, Յ35Å) by coupling chemical cross-linking with high-resolution mass spectrometry (MS) to derive peptide sequence information.…”

mentioning

confidence: 99%

“…Sequence identification of affinity captured cross-linked peptide pairs is achieved using Fourier transform ion cyclotron resonance (FT-ICR) MS coupled with Real-time Analysis Cross-link Technology (ReACT). The MS-labile bonds in PIR molecules allow for the controlled release of the cross-linked peptides within the mass spectrometer so that fragmentation of the peptide backbone can occur on each peptide individually (16,(18)(19)(20). The ReACT algorithm allows for the tandem identification of two cross-linked peptides by validating the PIR mass relationship (precursor ϭ mass peptide 1 ϩ mass peptide 2 ϩ mass reporter) of the original cross-linked species in real time.…”

mentioning

confidence: 99%

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Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

DeBlasio

Chavez

Alexander

et al. 2016

J Virol

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Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection-hallmarks of host-pathogen interactions-were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCEThe exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy. P hloem-limited viruses, such as poleroviruses, are among the most devastating, but least understood pathogens infecting plants due to the technical challenges encountered when studying pathogens that naturally replicate and move within intractable cell types (1). Mutagenesis studies performed with infectious clones have greatly enhanced our understanding of the domains within viral proteins that are re...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

DeBlasio

Chavez

Alexander

et al. 2016

J Virol

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Luteovirids

Boissinot¹,

Brault²,

Herrbach³

2020

Encyclopedia of Life Sciences

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Members of the Luteoviridae family, or luteovirids, are plant viruses that can infect a wide range of host plants, including many important crops such as cereals, potato, sugar beet, cucurbits, legumes and sugarcane. Their icosahedral virions contain a single positive‐sense ribonucleic acid genome in a capsid composed of two structural proteins. They are limited to phloem cells in host plants and are only transmitted by aphids in a circulative and nonpropagative mode with high specificity. More data are accumulating on plant–luteovirid relationships and in particular on the mechanism developed by the virus to overcome plant defence. The tight interaction between luteovirids, their aphid vector and their host plant are also extensively studied. Information from these studies together with a better understanding of the epidemiology of luteovirids will help to combat their detrimental effects on crops. Key Concepts Luteovirids have a highly compacted genome and use a diversity of translation mechanisms to express viral proteins. Function of the viral gene products is known, but their plant and aphid partners have not been fully identified. Luteovirids are confined to phloem tissue, but the causes of their phloem restriction are not completely deciphered. Luteovirids circulate in the vector aphid's body, without any replication, by crossing the gut and the accessory salivary gland epithelia. Epidemiology of luteovirids reveals a high complexity of mutual ecological interactions between virus, plant and vector, influenced by multiple biotic and abiotic factors. Control of luteoviral diseases includes cultural practices, insecticidal measures, natural and biotechnological plant resistance, and epidemiology‐based forecasting systems.

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Mass Spectrometry in Virological Sciences

Milewska

Ner‐Kluza

Dąbrowska

et al. 2019

Mass Spectrometry Reviews

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Virology, as a branch of the life sciences, discovered mass spectrometry (MS) to be the pivotal tool around two decades ago. The technique unveiled the complex network of interactions between the living world of pro-and eukaryotes and viruses, which delivered "a piece of bad news wrapped in protein" as defined by Peter Medawar, Nobel Prize Laureate, in 1960. However, MS is constantly evolving, and novel approaches allow for a better understanding of interactions in this micro-and nanoworld. Currently, we can investigate the interplay between the virus and the cell by analyzing proteomes, interactomes, virus-cell interactions, and search for the compounds that build viral structures. In addition, by using MS, it is possible to look at the cell from the broader perspective and determine the role of viral infection on the scale of the organism, for example, monitoring the crosstalk between infected tissues and the immune system. In such a way, MS became one of the major tools for the modern virology, allowing us to see the infection in the context of the whole cell or the organism.

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Cross-linking Measurements of the Potato leafroll virus Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Cited by 62 publications

References 79 publications

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

Luteovirids

Mass Spectrometry in Virological Sciences

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